To understand IFN resistance in vivo, we previously examined the dynamic responses to human IFN (hIFN)-alfa, -gamma and consensus IFN in the chimpanzee model. We showed that there is a defective response, particularly in the liver, to IFNs in HCV-infected chimpanzees, and this defect is possibly mediated through the activation of SOCS3. Further study on the inhibitory mechanism of IFN effector pathway by HCV infection in chimpanzee may provide novel insights into the clinical issue of nonresponse to IFN therapy. To further explore the mechanisms of IFN action and resistance in HCV patients, we compared hepatic gene expression in patients prior to and during peginterferon and ribavirin therapy. In the on-treatment group patients received either peginterferon alfa-2a alone or ribavirin for 72 hours and peginterferon-alfa 24 hours prior to biopsy. Patients were grouped into rapid responders (RR) and slow responders (SR). Pre-treatment biopsy specimens were obtained from a matched control group. Pre-treatment patients were grouped as RR or SR based on subsequent treatment response. Gene expression profiling was performed using Affymetrix microarray technology. Known ISGs were induced in treated patients. In the pre-treatment group, future slow responders (SR) had higher pretreatment ISG expression than rapid responders (RR). On treatment, RR and SR had similar absolute ISG expression but when corrected for baseline expression using the pre-treatment group, RR had marked induction of ISGs while SR showed up-regulation of IFN-inhibitory pathways. Patients pretreated with ribavirin had heightened induction of IFN-related genes as well as down-regulation of genes involved in IFN-inhibition and hepatic stellate cell (HSC) activation. These data suggest that ISG inducibility is important for treatment response and ribavirin may improve outcomes by enhancing hepatic gene responses to peginterferon. Collectively these mechanisms may provide a molecular basis for the improved efficacy of combination therapy.[unreadable] [unreadable] To further explore the mechanism of ribavirin action, we evaluated early viral kinetics, serum cytokine expression and viral mutagenesis during peginterferon treatment with or without ribavirin. 50 patients with genotype 1 infection were randomized to receive peginterferon alfa 2a with (Group A) or without (Group B) ribavirin for the first month of treatment. All patients then received a full 48 weeks of combination therapy. First and second phase viral kinetics were evaluated. Serum IP10, MIG1 and MCP1 levels were measured at time 0, 12 hours, day 3 and day 7. Viral sequencing of a 1772 nucleotide region spanning part of NS5A and NS5B was performed at day 0, 7 and 28 and mutation rates were calculated. First phase decline was similar between groups. Patients receiving ribavirin had more rapid second phase kinetics, but only in those patients with a good first phase decline (>0.5 log IU/ml) (median -0.72 vs -0.38 log/ml/week, p=0.039). Of the patients with a good first phase decline, 14/15 (93%) in Group A went on to have a good second phase slope (<0.3 log10IU/ml/week), compared to11/18 (61%) in Group B (p=0.046). The induction of serum IP10 at 12 hours was higher in patients receiving ribavirin than those receiving peginterferon alone (7.4 vs 3.8 fold, p=0.01), however similar to second phase slope, the difference in IP10 induction was more apparent in those with a good first phase decline. IP10 induction at 12 hrs correlated with first (p=0.0004, r2=040) and second phase (p=0.001, r2=0.40) kinetics in patients who received ribavirin, but not in those receiving peginterferon alone (phase 1 p=0.27, r2=0.05, phase 2 p=0.6, r2=0.013). IP10 induction at day 3 and 7 was also higher in patients receiving ribavirin, but only in those with good first phase kinetics. Results with MIG1 and MCP1 data were similar to those with IP10. Sequencing analysis in 19 patients (11 Group A, 8 Group B) revealed similar synonymous and non-synonymous mutation rates in patients receiving ribavirin as those receiving peginterferon alone. Early and sustained viral responses were similar between groups (EVR A 73% vs B 83%, SVR A 52% vs B 48%, p=NS). In this study, ribavirin improves early viral kinetics, but only in those patients with an initial response to peginterferon. The greater induction of interferon-stimulated cytokines and the correlation with viral kinetics in ribavirin-treated patients, suggest that the effect of ribavirin may be mediated through augmentation of interferon signaling.[unreadable] [unreadable] We also tested ribavirin in the infectious HCV cell culture systems. Similar to interferon-alfa, ribavirin potently inhibits JFH-1 infection of Huh7.5.1 cells in a dose-dependent manner, which spans the physiological concentration of ribavirin in vivo. Microarray analysis and subsequent quantitative PCR assays demonstrated that ribavirin treatment results in the induction of a specific set of interferon stimulated genes (ISGs) including IRF7, IRF9, and ISG15 which are known to play an important role in anti-HCV response. Upregulation of these antiviral genes by ribavirin is mediated by a novel mechanism different from those known to be associated with interferon action and intracellular double stranded RNA sensing pathways such as RIG-I and MDA5. RNA interference studies excluded the activation of the Toll-like receptor and NF-Kappa B pathways in the action of ribavirin. Additional experiments demonstrated the involvement of a short-lived transcriptional repressor whose activity is inhibited by ribavirin resulting in the upregulation of these antiviral genes. Our study suggests that ribavirin, acting via a novel innate mechanism, potentiates the antiviral effect of interferon-alfa in the treatment of hepatitis C. Understanding the mechanism of action of ribavirin is valuable in identifying novel antiviral molecules that could be used in combination with interferon.